virotype CSFV RT-PCR Kit

For detection of RNA from classical swine fever virus in samples from swine

Rapid and specific detection of RNA from classical swine fever virus

Easy-to-use single reaction mix containing controls

RT-PCR run time of less than 2 hours

Can be performed in one cycler run with other virotype RT-PCR assays

The virotype CSFV RT-PCR Kit allows the safe detection of RNA from classical swine fever virus (CSFV) in samples from pigs and wild boar. The kit contains all of the reagents for real-time RT-PCR in a single ready-to-use reaction mix. It can be used with individual or pooled serum, plasma, EDTA-stabilized blood, and tissue samples.

The virotype CSFV RT-PCR Kit is for veterinary use only.

In Germany: Registered in accordance with §17c of the German Law on Animal Diseases. Registration No.: FLI-B 517

In Germany: Registered in accordance with §17c of the German Law on Animal Diseases. Registration No.: FLI-B 517

Performance

virotype CSFV RT-PCR Kit detects RNA from classical swine fever virus. A validation study of 553 blood and tissue samples, with known CSFV status (295 positive and 258 negative; positive samples were provided by the EU reference laboratory CSFV, Hannover, National reference laboratory CSF, FLI Insel Riems, State laboratory; negative samples were provided by state and private laboratories), was carried out with the virotype CSFV RT-PCR Kit. Of 258 negative samples, 1 sample tested positive, providing a diagnostic specificity of > 99.5%. All positive samples tested positive, providing a diagnostic sensitivity of 100%. A subsequent study using an Epizone panel (kindly provided by Dr. Bernd Hoffmann, Friedrich-Loeffler-Institute) of non-CSFV pestiviruses was tested with the virotype CSFV RT-PCR Kit. All negative samples tested negative, providing a high diagnostic specificity (see the table Specificity of virotype CSFV RT-PCR Kit). Positive RNA samples of the Epizone panel of different relevant CSFV genotypes were tested with the virotype CSFV RT-PCR Kit alongside the CSF-1 in-house method (Hoffmann et al., 2005). All of the samples tested positive providing a high diagnostic sensitivity. A comparison of the CT values attained by the virotype CSFV RT-PCR Kit and the in-house CSF-1 method illustrates the virotype CSFV RT-PCR Kit is more sensitive (see the table Sensitivity of virotype CSFV RT-PCR Kit).

Specificity of virotype CSFV RT-PCR Kit

Pestivrus RNA samples

Origin

virotype CSFV RT-PCR result

BDV

Moredun

Negative

BDV

Rudolph

Negative

BDV

Gifhorn

Negative

BDV

Isard

Negative

BVDV-1

NADL

Negative

BVDV-1

Paplitz

Negative

BVDV-1

PI809

Negative

BVDV-1

NC3807-1251/1

Negative

BVDV-1

Egbert

Negative

BVDV-1

BO806-17

Negative

BVDV-1

BO807-3

Negative

BVDV-1

NC3087-8757

Negative

BVDV-2

8644

Negative

BVDV-2

Bure

Negative

BVDV-2

Walter

Negative

BVDV-2

PO1600

Negative

Pestivirus

Hobi

Negative

Pestivirus

Giraffe H138

Negative

A panel of non-CSFV pestiviruses was tested with the virotype CSFV RT-PCR Kit. All of the samples tested negative, providing a diagnostic specificity for this sample set of 100%.

Sensitivity of virotype CSFV RT-PCR Kit

Strain

Genotype

virotype CSFV CT

CSF-1 CT

C strain

1.1

27.45

31.21

Eystrup91

1.1

29.94

32.01

Alfort187

1.1

28.89

32.03

Koslov1128

1.2

27.98

31.64

Brescia

1.2

29.35

32.94

Scweiz II

2.1

29.34

31.07

Pader

2.1

29.66

30.94

Bergen

2.2

29.97

34.46

D4886/82/Ro

2.2

28.69

30.84

Uelzen

2.3

27.16

29.16

Spante

2.3

28.42

29.54

Congenital tremor

3.1

25.90

28.85

Kanagawa

3.4

27.25

30.65

RNA samples of the Epizone panel were tested with the virotype CSFV RT-PCR Kit alongside the CSF-1 in-house method. All of the samples tested positive. The virotype CSFV RT-PCR Kit sensitively detects all tested strains. The sensitivity of the virotype CSFV RT-PCR Kit, for the tested strains, is overall higher than the in-house CSF-1 method.

Principle

This is a real-time RT-PCR kit for the detection of RNA from classical swine fever virus. RT-PCR is based on the amplification of specific regions of the pathogen genome. It is a sensitive and specific tool for pathogen detection.

Real-time RT-PCR involves the reverse transcription of viral RNA and subsequent amplification of the cDNA by PCR. The amplified product is detected using fluorescent dyes. These are usually linked to oligonucleotide probes that bind specifically to the amplified product. Monitoring the fluorescence intensities during the PCR run (i.e., in real time) allows the detection of the accumulating product without the need to re-open the reaction tubes afterward.

Procedure

The kit contains a ready-to-use mix with all of the necessary reagents for the detection of CSFV RNA and the required positive and negative controls. With this kit, both reverse transcription and PCR are performed in one reaction tube, reducing the risk of contamination.

RNA is extracted from the sample material (serum, plasma, EDTA-stabilized blood, and tissue) using a suitable sample preparation kit. The extracted RNA is used for the RT-PCR, which is run on a thermal cycler.

Applications

The virotype CSFV RT-PCR Kit is for detection of RNA from classical swine fever virus (CSFV) in serum, plasma, EDTA-stabilized blood, and tissue samples from swine.